P38MAP kinase inhibitors

ABSTRACT

The present invention relates to a compound of formula (I): 
                         
or a pharmaceutically acceptable salt thereof, including all stereoisomers and tautomers, which is an inhibitor of p38 mitogen-activated protein kinase enzymes (referred to herein as p38 MAP kinase inhibitors), particularly the alpha and gamma kinase sub-types thereof, and its use in therapy, including in pharmaceutical combinations, especially in the treatment of inflammatory diseases, including inflammatory diseases of the lung, such as COPD.

This application is a continuation of application Ser. No. 13/262,266filed on Dec. 1, 2011, which is a National Stage application under 35U.S.C. 371 of PCT International Application No. PCT/GB2010/050575, filedApr. 1, 2010, which claims priority from Patent Application No. GB0905955.1, filed Apr. 6, 2009, and U.S. Provisional Application No.61/166,594 filed Apr. 3, 2009, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to a compound which is an inhibitor of p38mitogen-activated protein kinase enzymes (referred to herein as p38 MAPkinase inhibitors), particularly the alpha and gamma kinase sub-typesthereof, and its use in therapy, including in pharmaceuticalcombinations, especially in the treatment of inflammatory diseases,including inflammatory diseases of the lung, such as COPD.

BACKGROUND OF THE INVENTION

Four p38 MAPK isoforms (alpha, beta, gamma and delta respectively) havebeen identified, each displaying a tissue-specific expression pattern.The p38 MAPK alpha and beta isoforms are ubiquitously expressedthroughout the body and are found in many different cell types. The p38MAPK alpha and beta isoforms are inhibited by certain known smallmolecule p38 MAPK inhibitors. Earlier generations of compounds werehighly toxic due to the ubiquitous expression pattern of these isoformsand off-target effects of the compounds. More recent inhibitors areimproved to be highly selective for p38 MAPK alpha and beta isoforms andhave a wider safety margin.

Less is known about the p38 MAPK gamma and delta isoforms. Theseisoforms are expressed in specific tissues/cells (unlike the p38 alphaand p38 beta isoforms). The p38 MAPK-delta isoform is expressed more inthe pancreas, testes, lung, small intestine and kidney. It is alsoabundant in macrophages (Smith, S. J. (2006) Br. J. Pharmacol.149:393-404) and detectable in neutrophils, CD4+ T cells and endothelialcells (www.genecard.org, Karin, K. (1999) J. Immunol.). Very little isknown about the expression of p38 MAPK gamma but it is expressed more inbrain, skeletal muscle and heart, as well as in lymphocytes andmacrophages (www.genecard.org).

Selective small molecule inhibitors of p38 MAPK-gamma and -delta are notcurrently available, but one existing compound, BIRB 796, is known tohave pan-isoform inhibitory activity. The p38 gamma and p38 deltainhibition is observed at higher concentrations of the compound thanthose required to inhibit p38 MAPK alpha (Kuma, Y. (2005) J. Biol. Chem.280:19472-19479). BIRB 796 also impaired the phosphorylation of p38MAPKs or JNKs by the upstream kinase MKK6 or MKK4. Kuma discussed thepossibility that the conformational change caused by the binding of theinhibitor to the MAPK may affect the structure of both itsphosphorylation site and the docking site for the upstream activator,therefore impairing the phosphorylation of p38 MAPKs or JNKs.

p38 MAP kinase is believed to play a pivotal role in many of thesignalling pathways that are involved in initiating and maintainingchronic, persistent inflammation in human disease, for example, severeasthma and COPD. There is now abundant literature which demonstratesthat p38 MAP kinase is activated by a range of pro-inflammatorycytokines and that its activation results in the recruitment and releaseof further pro-inflammatory cytokines. Indeed, data from some clinicalstudies demonstrate beneficial changes in disease activity in patientsduring treatment with p38 MAP kinase inhibitors. For instance Smith, S.J. (2006) Br. J. Pharmacol. 149:393-404 describes the inhibitory effectof p38 MAP kinase inhibitors on TNFα (but not IL-8) release from humanPBMCs. Use of inhibitors of p38 MAP kinase in the treatment of chronicobstructive pulmonary disease (COPD) is proposed. Small moleculeinhibitors targeted to p38 MAPKα/β have proved to be effective inreducing various parameters of inflammation in cells and tissuesobtained from patients with COPD, who are generally corticosteroidinsensitive, (Smith, S. J. (2006) Br. J. Pharmacol. 149:393-404) and invivo animal models (Underwood, D. C. et al. (2000) 279:895-902; Nath, P.et al. (2006) Eur. J. Pharmacol. 544:160-167; Medicherla S. et al.(2008) J. Pharm. Exp. Ther. 324:921-929). Irusen and colleagues alsosuggested the possibility of involvement of p38 MAPK α/β oncorticosteroid insensitivity via reduction of binding affinity ofglucocorticoid receptor (GR) in nuclei (Irusen, E. et al., (2002) J.Allergy Clin. Immunol., 109:649-657). Clinical experience with a rangeof p38 MAP kinase inhibitors, including AMG548, BIRB 796, VX702, SCIO469and SCIO0323 is described in Lee et al. (2005) Current Med. Chem. 12,:2979-2994.

COPD is a condition in which the underlying inflammation has beenreported to be substantially resistant to the anti-inflammatory effectsof inhaled corticosteroids. Consequently, an effective strategy fortreating COPD may well be to develop an intervention which both hasinherent anti-inflammatory effects and is able to increase thesensitivity of lung tissues from COPD patients to inhaledcorticosteroids. The recent publication of Mercado et al. (2007)American Thoracic Society Abstract A56) demonstrates that silencing p38gamma has the potential to restore sensitivity to corticosteroids. Thusthere may be a “two pronged” benefit to the use of a p38 MAP kinaseinhibitor for the treatment of COPD and severe asthma.

There is now a substantial body of evidence which strongly implicatesthe role of respiratory viral infections in initiating exacerbations inpatients suffering from asthma and/or COPD. Exacerbations require anincrease in treatment intensity to re-establish control of diseasesymptomology. If severe, exacerbations may well result in thehospitalisation or, at its most extreme, the death of patients. Thoseviruses which are commonly associated with exacerbations includerhinovirus, influenza and respiratory syncitial virus. The cellularresponses to these viruses are now known to include the up-regulation ofICAM1 (intercellular adhesion molecule 1) and release of cytokines, aswell as the replication of virus particles. There has been someinvestigation into the effect of p38 MAP kinase inhibitors on theseviral responses and some reports suggest that protective effects can bedetected with p38 MAP kinase inhibitors. In particular, some reportssuggest that inhibition of virus-induced release of IL-8 can be achievedin vitro with the known compound, SB203580. It is noteworthy that theassessment of an agent's efficacy to reduce rhinovirus-inducedinflammation and virus replication in preclinical in vivo models remainsa significant challenge. However, there are well-established in vivomodels of influenza in the mouse and parainfluenza in the guinea pig.

The major obstacle hindering the utility of p38 MAP kinase inhibitors inthe treatment of human chronic inflammatory diseases has been thetoxicity observed in patients. This has been sufficiently severe toresult in the withdrawal from clinical development of many of thecompounds progressed, including all those specially mentioned above.

There remains a need to identify and develop new compoundstherapeutically useful as p38 MAP kinase inhibitors which have improvedtherapeutic potential, in particular which are more efficacious, longeracting and/or less toxic at the relevant therapeutic dose. An objectiveof the present invention is to provide a compound which inhibits p38 MAPkinase, for example with certain sub-type specificity, which shows goodanti-inflammatory potential.

SUMMARY OF THE INVENTION

According to the invention, there is provided a compound of formula (1)

or a pharmaceutically acceptable salt or solvate thereof, including allstereoisomers and tautomers thereof.

The systematic name of the compound of formula (1) isN-(4-(4-(3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results from treatment with Compound (1) in a cigarettesmoke model. Data for cell (neutrophils) numbers are shown as themean±SEM.

FIG. 2 shows results from treatment with Compound (1) in a cigarettesmoke model. Data for cell (activated alveolar macrophages) numbers areshown as the mean±SEM.

FIG. 3 shows the effect of Compound 1 as monotherapy in a ovalbuminchallenge/parainfluenza infection model.

FIG. 4 shows the effect of Compound 1, when administered in combinationwith fluticasone propionate, in a ovalbumin challenge/parainfluenzainfection model.

DETAILED DESCRIPTION OF THE INVENTION

Examples of salts of compound (1) include all pharmaceuticallyacceptable salts, such as, without limitation, acid addition salts ofstrong mineral acids such as HCl and HBr salts and addition salts ofstrong organic acids such as a methansulfonic acid salt.

As employed herein below the definition of a compound of formula (1) isintended to include salts, solvates, and all tautomers of said compound,unless the context specifically indicates otherwise. Examples ofsolvates include hydrates.

The invention provided herein extends to prodrugs of the compound offormula (1), that is to say compounds which break down and/or aremetabolised in vivo to provide an active compound of formula (1).General examples of prodrugs include simple esters, and other esterssuch as mixed carbonate esters, carbamates, glycosides, ethers, acetalsand ketals.

In a further aspect of the invention there is provided one or moremetabolites of the compound of formula (1), in particular a metabolitethat retains one or more of the therapeutic activities of the compoundof formula (1). A metabolite, as employed herein, is a compound that isproduced in vivo from the metabolism of the compound of formula (1),such as, without limitation, oxidative metabolites and/or metabolitesgenerated, for example, from O-dealkylation.

The compounds of the disclosure include those where the atom specifiedis a naturally occurring or non-naturally occurring isotope. In oneembodiment the isotope is a stable isotope. Thus the compounds of thedisclosure include, for example deuterium containing compounds and thelike.

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

An example of a route of preparing the compound of formula (1) is shownbelow in scheme 1:

Thus the compound of formula (1) can be prepared by a process comprisingreacting a compound of formula (8):

with a compound of formula (A):

where LG₁ is a leaving group for example halogen, such as chloro.

The reaction is suitably carried out in the presence of a base (e.g.N,N-diisopropylethylamine).

Compounds of formula (8) can be prepared by reacting a compound offormula (7)

with a compound of formula (6):

and a compound of formula (B):

wherein LG₂ and LG₃ each independently represent leaving groups (e.g.LG₂ is Cl₃CO— and LG₃ is Cl. Alternatively both LG₂ and LG₃ may be thesame, for example LG₂ and LG₃ both represent imidazolyl or halogen suchas chloro).

The reaction is suitably carried out in an aprotic solvent (e.g.dichloromethane), using appropriate protecting groups for chemicallysensitive groups.

A compound of formula (6) may be prepared by reaction of a compound offormula (4a):

wherein P¹ is a suitable amine protective group and LG₄ represents aleaving group such as chloro, with a compound of formula (C)

for example in the presence of a suitable catalyst such as a homogeneouspalladium catalyst, followed by removal of the amine protective groups.For example, when the protective groups are tert-butoxycarbonyl, theirremoval can be achieved by treatment with TFA in dichloromethane.

Compounds of formula (4a) may be prepared by protecting a compound offormula (3a):

for example employing di-tert-butyl dicarbonate and DMAP, wherein LG₄represents a leaving group, such as chloro.

Compounds of formula (3a) can be prepared by reacting a compound offormula (2):

with a compound of formula (D):

wherein LG₄ represents a leaving group, for example a halogen atom, suchas chlorine, as per the compound of formula (3), under appropriatereaction conditions, for example in the presence of a strong base suchas potassium tert-butoxide and N-methylpyrrolidone(1-methylpyrrolidin-2-one).

The compound of formula (1) can also be prepared by the processrepresented in Scheme 2:

Thus the compound of formula (1) can be prepared by reacting a compoundof formula (7):

with a compound of formula (12):

for example in the presence of a suitable coupling agent, such as1,1-carbonyl-diimidazole. The compound of formula (12) can be preparedby the reduction of a compound of formula (11):

The reduction may be performed under hydrogenation conditions over asuitable catalyst such as palladium on carbon, or alternatively may beperformed chemically using an appropriate reducing agent, such as ironpowder in acetic acid.

The compound of formula (11) can be prepared by reacting a compound offormula (10)

with a compound of formula (A):

wherein LG₁ is represents a leaving group, as defined above.

The reaction may be performed in a suitable solvent, for exampledichloromethane in the presence of a suitable non-nucleophilic base suchas N,N-diisopropylethylamine.

The compounds of formula (10) can be prepared by reacting a compound offormula (9):

with a compound of formula (E):

wherein the group NR¹R² is an amine or a suitably protected derivativethereof, for example in the presence of a base such as sodium hydrideand suitable solvent such as DMF. Where NR¹R² is not —NH₂, it will beappreciated that a suitable deprotection step must be included toprovide the compound of formula (10).

Compounds of formulae (2), (7), (9), (A), (B), (C), (D) and (E) areeither commercially available, or are known, or are novel and can bereadily prepared by conventional methods. See for example Regan, J. etal.; J. Med. Chem., 2003, 46, 4676-4686, WO00/043384, WO2007/087448 andWO2007/089512.

Protective groups may be required to protect chemically sensitive groupsduring one or more of the reactions described above, to ensure that theprocess can be carried out and/or is efficient. Thus if desired ornecessary, intermediate compounds may be protected by the use ofconventional protective groups. Protective groups and the means fortheir removal are described in “Protective Groups in Organic Synthesis”,by Theodora W. Greene and Peter G. M. Wuts, published by John Wiley &Sons Inc; 4^(th) Rev Ed., 2006, ISBN-10: 0471697540. Novel intermediatesas described herein form an aspect of the invention.

The compound of formula (1) is a p38 MAP kinase inhibitor and in oneaspect the compound is useful in treatment of diseases, for example COPDand/or asthma.

Surprisingly, the compound seems to have a long duration of actionand/or persistence of action in comparison to other known p38 MAP kinaseinhibitors such as BIRB796.

In one embodiment the compound of formula (1) is a p38 MAPK alpha and/orgamma sub-type inhibitor.

Persistence of action as used herein is related to the dissociation rateor dissociation constant of the compound from the target (such as areceptor). A low dissociation rate may lead to persistence.

A low dissociation rate in combination with a high association ratetends to provide potent therapeutic entities.

The compound of formula (1) may be potent in vivo.

Typically, the prior art compounds developed to date have been intendedfor oral administration. This strategy involves optimizing compoundswhich achieve their duration of action by an appropriate pharmacokineticprofile. This ensures that there is a sufficient drug concentrationestablished and maintained after and between doses to provide clinicalbenefit. The inevitable consequence of this approach is that all bodytissues, especially liver and gut, are likely to be exposed totherapeutically active concentrations of the drug, whether or not theyare adversely affected by the disease being treated.

An alternative strategy is to design treatment approaches in which thedrug is dosed directly to the inflamed organ (topical therapy). Whilethis approach is not suitable for treating all chronic inflammatorydiseases, it has been extensively exploited in lung diseases (asthma,COPD), skin diseases (atopic dermatitis and psoriasis), nasal diseases(allergic rhinitis) and gastrointestinal diseases (ulcerative colitis).

In topical therapy, efficacy can be achieved either by (i) ensuring thatthe drug has a sustained duration of action and is retained in therelevant organ to minimize the risks of systemic toxicity or (ii)producing a formulation which generates a “reservoir” of the active drugwhich is available to sustain the drug's desired effects. Approach (i)is exemplified by the anticholinergic drug tiotropium (Spiriva), whichis administered topically to the lung as a treatment for COPD, and whichhas an exceptionally high affinity for its target receptor resulting ina very slow off rate and a consequent sustained duration of action.

In one aspect of the disclosure the compound of formula (1) isparticularly suitable for topical delivery, such as topical delivery tothe lungs, in particular for the treatment of respiratory disease, forexample chronic respiratory disease such as COPD and/or asthma.

In one embodiment the compound of formula (1) is suitable forsensitizing patients to treatment with a corticosteroid who have becomerefractory to such treatment regimens.

The compound of formula (1) may also be useful for the treatment ofrheumatoid arthritis.

The compound of formula (1) may have antiviral properties, for examplethe ability to prevent infection of cells (such as respiratoryepithelial cells) with a picornavirus, in particular a rhinovirus,influenza or respiratory synctial virus.

Thus the compound is thought to be an antiviral agent, in particularsuitable for the prevention, treatment or amelioration of picornavirusinfections, such as rhinovirus infection, influenza or respiratorysyncitial virus.

In one embodiment the compound of formula (1) is able to reduceinflammation induced by viral infection, such as rhinovirus infectionand in particular viral infections that result in the release ofcytokines such as IL-8, especially in vivo. This activity may, forexample be tested in vitro employing a rhinovirus induced IL-8 assay asdescribed in the Examples herein.

In one embodiment the compound of formula (1) is able to reduce ICAM1expression induced by rhinovirus, especially in vivo. ICAM1 is thereceptor mechanism used by so-called major groove rhinovirus serotypesto infect cells. This activity may be measured, for example by a methoddescribed in the Examples herein.

It is expected that the above properties render the compound of formula(1) particularly suitable for use in the treatment and/or prophylaxis ofexacerbations, in particular viral exacerbations, in patients with oneor more the following chronic conditions such as congestive heartfailure, COPD, asthma, diabetes, cancer and/or in immunosuppressedpatients, for example post-organ transplant.

In particular, the compound of formula (1) may be useful in thetreatment of one or more respiratory disorders including COPD (includingchronic bronchitis and emphysema), asthma, paediatric asthma, cysticfibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis,rhinitis, sinusitis, especially asthma, and COPD (including chronicbronchitis and emphysema).

The compound of formula (1) may also be useful in the treatment of oneor more conditions which may be treated by topical or local therapyincluding allergic conjunctivitis, conjunctivitis, allergic dermatitis,contact dermatitis, psoriasis, ulcerative colitis, inflamed jointssecondary to rheumatoid arthritis or osteoarthritis.

It is also expected that the compound of formula (1) may be useful inthe treatment of certain other conditions including rheumatoidarthritis, pancreatitis, cachexia, inhibition of the growth andmetastasis of tumours including non-small cell lung carcinoma, breastcarcinoma, gastric carcinoma, colorectal carcinomas and malignantmelanoma.

The compound of formula (1) may also re-sensitise the patient'scondition to treatment with a corticosteroid, when the patient'scondition has become refractory to the same.

Furthermore, the present invention provides a pharmaceutical compositioncomprising a compound according to the disclosure optionally incombination with one or more pharmaceutically acceptable diluents orcarriers.

Diluents and carriers may include those suitable for parenteral, oral,topical, mucosal and rectal administration.

As mentioned above, such compositions may be prepared e.g. forparenteral, subcutaneous, intramuscular, intravenous, intra-articular orperi-articular administration, particularly in the form of liquidsolutions or suspensions; for oral administration, particularly in theform of tablets or capsules; for topical e.g. pulmonary or intranasaladministration, particularly in the form of powders, nasal drops oraerosols and transdermal administration; for mucosal administration e.g.to buccal, sublingual or vaginal mucosa, and for rectal administratione.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). Formulations for parenteral administration may contain asexcipients sterile water or saline, alkylene glycols such as propyleneglycol, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. Formulationsfor nasal administration may be solid and may contain excipients, forexample, lactose or dextran, or may be aqueous or oily solutions for usein the form of nasal drops or metered spray. For buccal administrationtypical excipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or morephysiologically compatible carriers and/or excipients and may be insolid or liquid form. Tablets and capsules may be prepared with bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpoly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch,calcium phosphate, sorbitol, or glycine; lubricants, such as magnesiumstearate, talc, polyethylene glycol, or silica; and surfactants, such assodium lauryl sulfate. Liquid compositions may contain conventionaladditives such as suspending agents, for example sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or ediblefats; emulsifying agents such as lecithin, or acacia; vegetable oilssuch as almond oil, coconut oil, cod liver oil, or peanut oil;preservatives such as butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Liquid compositions may be encapsulated in, forexample, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsulesand soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60%concentration of gelatin, about a 20% to 30% concentration ofplasticizer (such as glycerin, sorbitol or propylene glycol) and about a30% to 40% concentration of water. Other materials such aspreservatives, dyes, opacifiers and flavours also may be present. Theliquid fill material comprises a solid drug that has been dissolved,solubilized or dispersed (with suspending agents such as beeswax,hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug invehicles or combinations of vehicles such as mineral oil, vegetableoils, triglycerides, glycols, polyols and surface-active agents.

Suitably the compound of formula (1) is administered topically to thelung. Hence we provide according to the invention a pharmaceuticalcomposition comprising a compound of the disclosure optionally incombination with one or more topically acceptable diluents or carriers.Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoro methane (propellant 114), anddichlorodifluoromethane (propellant 12). Suitable HFC propellantsinclude tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).The propellant typically comprises 40% to 99.5% e.g. 40% to 90% byweight of the total inhalation composition. The formulation may compriseexcipients including co-solvents (e.g. ethanol) and surfactants (e.g.lecithin, sorbitan trioleate and the like). Aerosol formulations arepackaged in canisters and a suitable dose is delivered by means of ametering valve (e.g. as supplied by Bespak, Valois or 3M).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser. Topical administrationto the lung may also be achieved by use of a dry-powder formulation. Adry powder formulation will contain the compound of the disclosure infinely divided form, typically with a mass mean aerodynamic diameter(MMAD) of 1-10 microns. The formulation will typically contain atopically acceptable diluent such as lactose, usually of large particlesize e.g. an MMAD of 100 μm or more. Examples of dry powder deliverysystems include SPINHALER, DISKHALER, TURBOHALER, DISKUS and CLICKHALER.

The compound of formula (1) has therapeutic activity. In a furtheraspect, the present invention provides a compound of the disclosure foruse as a medicament. Thus, in a further aspect, the present inventionprovides a compound as described herein for use in the treatment of oneor more of the above mentioned conditions.

In a further aspect, the present invention provides use of a compound asdescribed herein for the manufacture of a medicament for the treatmentof one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method oftreatment of one or more of the above mentioned conditions whichcomprises administering to a subject an effective amount of a compoundof the disclosure or a pharmaceutical composition comprising thecompound.

The word “treatment” is intended to embrace prophylaxis as well astherapeutic treatment. A compound of the disclosure may also beadministered in combination with one or more other active ingredientse.g. active ingredients suitable for treating the above mentionedconditions. For example possible combinations for treatment ofrespiratory disorders include combinations with steroids (e.g.budesonide, beclomethasone dipropionate, fluticasone propionate,mometasone furoate, fluticasone furoate), beta agonists (e.g.terbutaline, salbutamol, salmeterol, formoterol) and/or xanthines (e.g.theophylline). Other suitable actives include anticholinergics, such astiotropium and anti-viral agents such as, but not limited to, zanamiviror oseltamivir, for example as the phosphate. Other anti-viral agentsinclude peramivir and laninamivir.

The data generated below in relation to the antiviral properties of thecompound of formula (1) leads the inventors to believe that otherantiviral therapies would be useful in the treatment or prevention ofexacerbations of patients with respiratory disease such as COPD and/orasthma and/or one or more of the indications listed above. Thus in oneaspect there is provided the use of an anti-viral therapy, such as, butnot limited to, zanamavir or oseltamivir (for example oseltamivirphosphate), in the treatment or prevention of respiratory viralinfections in patients with chronic conditions such as congestive heartfailure, diabetes, cancer, or in immunosuppressed patients, for examplepost-organ transplant.

ABBREVIATIONS

AcOH glacial acetic acid

Aq aqueous

Ac acetyl

ATP adenosine-5′-triphosphate

BALF bronchoalveolae lavage fluid

Br broad

BSA bovine serum albumin

CatCart® catalytic cartridge

CDI 1,1-carbonyl-diimidazole

COPD chronic obstructive pulmonary disease

D doublet

Pd₂(dba)₃ Tris(dibenzylideneacetone)dipalladium

DCM dichloromethane

DIAD diisopropylazadicarboxylate

DIBAL-H diisobutylaluminium hydride

DMAP N,N-dimethylpyridin-4-amine

DIPEA N,N-diisopropylethylamine

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

ELISA enzyme immunosorbent assay

EtOAc ethyl acetate

FCS foetal calf serum

FRET fluorescence resonance energy transfer

HEPES 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid

Hr hour(s)

HRP horseradish peroxidase

HRV human rhinovirus

ICAM1 intercellular adhesion molecule 1

IgG immunogloblin G

IL-8 interleukin 8

JNK c-Jun N-terminal kinase

LPS lipopolysaccharide

MAPK mitogen protein activated protein kinase

MAPKAP-K2 mitogen-activated protein kinase-activated protein kinase 2

MeOH methanol

Min minute(s)

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NMP N-methylpyrrolidone (1-methylpyrrolidin-2-one)

NSE no significant effect

OD optical density

PBMC peripheral blood mononuclear cell

PBS phosphate buffered saline

PMA phorbol myristate acetate

PPh₃ triphenylphosphine

RSV respiratory syncitial virus

RT room temperature

RP HPLC reverse phase high performance liquid chromatography

S singlet

SCX solid supported cation exchange

SDS sodium dodecyl sulfate

SiO₂ silica gel

T triplet

TCID₅₀ 50% tissue culture infection dose

TFA trifluoroacetic acid

THF tetrahydrofuran

TMB 3,3″,5,5″-tetramethylbenzidine

TNFα tumor necrosis factor alpha

XantPhos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

General Procedures

All starting materials and solvents were either obtained from commercialsources or prepared according to the literature citation. Hydrogenationswere preformed on a Thales H-cube flow reactor under the conditionsstated. Organic solutions were routinely dried over magnesium sulfate.SCX was purchased with Supelco and treated with 1M aqueous HCl prior touse. The reaction mixture to be purified was first diluted with MeOH andmade acidic with a few drops of AcOH. This solution was loaded directlyonto the SCX and washed with MeOH. The desired material was then elutedby washing with 1% NH₃ in MeOH. Column chromatography was performed onSilicycle pre-packed silica (230-400 mesh, 40-63 μM) cartridges usingthe amount indicated.

Preparative Reverse Phase High Performance Liquid Chromatography:

Agilent Scalar column C18, 5 μm (21.2×50 mm), flow rate 28 mL/mineluting with a H₂O-MeCN gradient containing 0.1% v/v formic acid over 10mins using UV detection at 215 and 254 nm. Gradient information: 0.0-0.5min: 95% H₂O-5% MeCN; 0.5-7.0 min; Ramped from 95% H₂O-5% MeCN to 5%H₂O-95% MeCN; 7.0-7.9 min: Held at 5% H₂O-95% MeCN; 7.9-8.0 min:Returned to 95% H₂O-5% MeCN; 8.0-10.0 min: Held at 95% H₂O-5% MeCN.

Analytical Methods

Reverse Phase High Performance Liquid Chromatography:

Agilent Scalar column C18, 5 μm (4.6×50 mm) or Waters XBridge C18, 5 μm(4.6×50 mm) flow rate 2.5 mL/min eluting with a H₂O-MeCN gradientcontaining 0.1% v/v formic acid over 7 min employing UV detection at 215and 254 nm. Gradient information: 0.0-0.1 min: 95% H₂O-5% MeCN; 0.1-5.0min; Ramped from 95% H₂O-5% MeCN to 5% H₂O-95% MeCN; 5.0-5.5 min: Heldat 5% H₂O-95% MeCN; 5.5-5.6 min: Held at 5% H₂O-95% MeCN, flow rateincreased to 3.5 ml/min; 5.6-6.6 min: Held at 5% H₂O-95% MeCN, flow rate3.5 ml/min; 6.6-6.75 min: Returned to 95% H₂O-5% MeCN, flow rate 3.5ml/min; 6.75-6.9 min: Held at 95% H₂O-5% MeCN, flow rate 3.5 ml/min;6.9-7.0 min: Held at 95% H₂O-5% MeCN, flow rate reduced to 2.5 ml/min.

¹H NMR Spectroscopy:

Bruker Avance III 400 MHz using residual undeuterated solvent asreference

Experimental Procedures for Scheme 14-(2-Chloropyridin-4-yloxy)naphthalen-1-amine (3)

To a stirred solution of 2-chloro-4-fluoropyridine (1.261 g, 9.58 mmol)and 4-amino-1-naphthol hydrochloride (2) (750 mg, 3.83 mmol) in NMP (40mL), at −20° C., was added potassium tert-butoxide (1.290 g, 11.50mmol). The reaction mixture was allowed to warm to RT. After 2.5 hr, thereaction mixture was diluted with water (100 mL) and extracted withEtOAc (100 mL then 2×80 mL). The combined organic extracts were washedwith brine (150 mL), dried and evaporated in vacuo. The crude productwas subjected to SCX capture and release eluting with 1% NH₃ in MeOHsolution and the solvent was removed in vacuo to give4-(2-chloropyridin-4-yloxy)naphthalen-1-amine (3) (1.019 g, 92%) as abrown solid: m/z 271 (M+H)⁺ (ES⁺).

4-(2-Chloropyridin-4-yloxy)naphthalen-1-N,N-di-tert-butylcarbamate (4)

To a stirred solution of 4-(2-chloropyridin-4-yloxy)naphthalen-1-amine(3) (1.019 g, 3.76 mmol) in THF (30 mL) at 0° C. was added DMAP (0.034g, 0.282 mmol) and then di-tert-butyl dicarbonate (0.904 g, 4.14 mmol).The reaction mixture was stirred at 0° C. for 30 min, and then allowedto warm to RT. After 1.5 hr, the reaction mixture was cooled to 0° C.,and further di-tert-butyl dicarbonate (0.904 g, 4.14 mmol) was added.The resulting mixture was stirred at 0° C. for 15 min and then at RT.After 16 hr the reaction mixture was diluted with water (40 mL) andextracted with EtOAc (2×40 mL). The combined organic extracts werewashed with brine (75 mL), dried and evaporated in vacuo. The crudematerial was purified by flash column chromatography (SiO₂; 80 g)eluting with 0 to 40% EtOAc in iso-hexane to give4-(2-chloropyridin-4-yloxy)naphthalen-1-N,N-di-tert-butylcarbamate (4)(0.892 g, 48%) as a purple solid: m/z 471 (M+H)⁺ (ES⁺).

tert-Butyl4-(4-(N,N-di-tert-butylcarbamyl)naphthalen-1-yloxy)pyridin-2-ylcarbamate(5)

A mixture of4-(2-chloropyridin-4-yloxy)naphthalen-1-N,N-di-tert-butylcarbamate (4)(0.892 g, 1.894 mmol), tert-butyl carbamate (0.666 g, 5.68 mmol),caesium carbonate (0.926 g, 2.84 mmol), Pd₂(dba)₃ (0.043 g, 0.047 mmol)and XantPhos (0.055 g, 0.095 mmol) was suspended in THF (10 mL). Thereaction mixture was purged thoroughly with nitrogen, and then heated atreflux. After 15 hr the mixture was cooled to RT, diluted with water (35mL) and extracted with EtOAc (35 mL, 25 mL). The combined organicextracts were washed with brine (50 mL), dried and evaporated in vacuo.The crude material was purified by flash column chromatography (SiO₂; 80g) eluting with 0 to 30% EtOAc in iso-hexane to give tert-butyl4-(4-(N,N-di-tert-butylcarbamyl)naphthalen-1-yloxy)pyridin-2-ylcarbamate(5) (289 mg, 28%) as a white solid: m/z 552 (M+H)⁺ (ES⁺).

4-(4-Aminonaphthalen-1-yloxy)pyridin-2-amine (6)

To a stirred solution of tert-butyl4-(4-(N,N-di-tert-butylcarbamyl)naphthalen-1-yloxy)pyridin-2-ylcarbamate(5) (289 mg, 0.524 mmol) in DCM (8 mL), at 0° C., was added TFA (4 mL).The resulting mixture was stirred while slowly warming to RT. After 5hr, the volatiles were removed in vacuo and the residue was taken up inMeOH (5 mL) and subjected to SCX capture and release eluting with 1% NH₃in MeOH solution. The solvent was removed in vacuo to afford4-(4-aminonaphthalen-1-yloxy)pyridin-2-amine (6) (116 mg, 85%) as abrown-orange oil: m/z 252 (M+H)⁺ (ES⁺).

1-(4-(2-aminopyridin-4-yloxy)naphthalen-1-yl)-3-(3-tert-butyl-1-D-tolyl-1H-pyrazol-5-yl)urea

A saturated solution of NaHCO₃ (14 mL) was added to a solution of5-aminopyrazole (7) (0.206 g, 0.900 mmol) in DCM (20 mL). The mixturewas stirred vigorously, cooled to 0° C. and trichloromethylchloroformate(0.326 mL, 2.70 mmol) was added in one portion. The reaction mixture wasstirred vigorously at 0° C. for a further 80 min. The layers wereseparated and the organic layer was dried, evaporated in vacuo and theresulting orange oil was dried further for 30 min under high vacuum. Theisolated isocyanate was then taken up into THF (6 mL) and kept undernitrogen at 0° C.

To a stirred solution of 4-(4-aminonaphthalen-1-yloxy)pyridin-2-amine(6) (116 mg, 0.462 mmol) and DIPEA (241 μl, 1.385 mmol) in THF (3 mL),at 0° C., was added an aliquot of the isocyanate solution prepared above(2 mL, 0.300 mmol). The resulting mixture was stirred while slowlywarming to RT. Additional aliquots of the isocyanate solution in THFwere added to the reaction mixture after 1.5 hr, (1 mL, 0.150 mmol) andafter a further 3.5 hr (0.5 mL, 0.075 mmol). After 20 hr water (30 mL)was added and the mixture was extracted with EtOAc (2×30 mL). Thecombined organic extracts were washed with brine (50 mL), dried andevaporated in vacuo. The crude material was purified by flash columnchromatography (SiO₂; 12 g) eluting with 25 to 100% [5% MeOH in EtOAc]in iso-hexane to furnish1-(4-(2-aminopyridin-4-yloxy)naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea(8) (127 mg, 49%) as a brown oil: m/z 507 (M+H)⁺ (ES⁺).

N-(4-(4-(3-(3-tert-Butyl-1-D-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(1)

Methoxyacetyl chloride (64.2 μl, 0.703 mmol) was added slowly to amixture of1-(4-(2-aminopyridin-4-yloxy)naphthalen-1-yl)-3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)urea (8) (89 mg, 0.176 mmol) and DIPEA (153 μl, 0.878 mmol) in THF (5mL). The reaction mixture was stirred at 0° C. for a further 20 min andthen warmed to RT. After 2.5 hr, the reaction was quenched by theaddition of a solution of 1% NH₃ in MeOH (3 mL), and the resultingmixture stirred for a further 45 min. The volatiles were removed invacuo and the residue was dissolved in a mixture of MeOH (5 mL) and AcOH(2 mL) and subjected to SCX capture and release eluting with 1% NH₃ inMeOH solution. The crude material was purified by flash columnchromatography (SiO₂; 12 g) eluting with 0 to 60% [5% MeOH in EtOAc] iniso-hexane to giveN-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(1) (62 mg, 61%) as a white solid: m/z 579 (M+H)⁺ (ES⁺). ¹H NMR (400MHz, DMSO-d₆) δ: 1.29 (9H, s), 2.40 (3H, s), 3.31 (3H, s), 3.99 (2H, s),6.41 (1H, s), 6.70 (1H, dd), 7.32-7.40 (3H, m), 7.44-7.48 (2H, m),7.55-7.61 (1H, m), 7.63-7.67 (2H, m), 7.84 (1H, dd), 7.97 (1H, d), 8.09(1H, d), 8.19 (1H, d), 8.79 (1H, s), 9.13 (1H, s), 10.02 (1H, s).

Experimental Procedures for Scheme 24-(4-Nitronaphthalen-1-yloxy)pyridin-2-amine (10)

To a stirred solution of 2-aminopyridine-4-ol (23.0 g, 209 mmol) in DMF(200 mL) at 0° C., under nitrogen was added NaH (60% dispersion inmineral oil, 9.21 g, 230 mmol) in portions over 30 mins. The mixture waswarmed to RT and after 1 hr cooled to 0° C. and a solution of1-fluoro-4-nitronaphthylene (9) (40.0 g, 209 mmol) in DMF (100 mL) wasadded. The reaction mixture was allowed to warm to RT and after 1.5 hrit was diluted with water (1 L) and extracted with EtOAc (3×500 mL). Theorganic extracts were combined and washed with water (3×700 mL) andbrine (500 mL). A yellow precipitate separated which was collected byfiltration and washed with water (500 mL) and dried in vacuo overnight.The organic layer was dried (MgSO₄), filtered and evaporated in vacuo.The residue was triturated with a mixture of acetonitrile (20 mL) andether (200 mL) to give a red solid which was washed with isohexane (200mL). The supernatant liquor from the trituration was evaporated in vacuoand the residue was purified by flash column chromatography (SiO₂, 120g, 30-100% EtOAc in isohexane, gradient elution). This product wascombined with the two solids isolated previously, taken up into MeOH(500 mL) and evaporated in vacuo to afford4-(4-nitronaphthalen-1-yloxy)pyridin-2-amine (10) (40.1 g, 65%) as ayellow solid: m/z 282 (M+H)⁺ (ES⁺).

2-Methoxy-N-(4-(4-nitronaphthalen-1-yloxy)pyridin-2-yl)acetamide (11)

To a stirred solution of 4-(4-nitronaphthalen-1-yloxy)pyridin-2-amine(10) (40.0 g, 135 mmol) and DIPEA (48.1 mL, 270 mmol) in DCM (600 mL) at0° C., under nitrogen, was added dropwise methoxyacetyl chloride (18.53ml, 203 mmol). The mixture was warmed to RT and after 1 hr a solution ofammonia (100 mL, 7 M in MeOH) was added and stirring continued for 30min, during which time a precipitate formed. The mixture was evaporatedin vacuo and water (1 L) was added to the residue to provide a redsuspension. Glacial acetic acid was added dropwise until a yellow colourpersisted (˜5 mL). The solid was collected by filtration and washed withwater (300 mL) to afford2-methoxy-N-(4-(4-nitronaphthalen-1-yloxy)pyridin-2-yl)acetamide (11)(44.1 g, 90%) as a yellow solid: m/z 354 (M+H)⁺ (ES⁺).

N-(4-(4-Aminonaphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide (12)

A stirred suspension of2-methoxy-N-(4-(4-nitronaphthalen-1-yloxy)pyridin-2-yl)acetamide (11)(44.0 g, 125 mmol) and iron powder (41.7 g, 747 mmol) in acetic acid(300 mL) was heated at 45° C. After 3 hr the mixture was cooled to RTand poured slowly and cautiously onto solid Na₂CO₃ (200 g). The mixtureeffervesced vigorously. The mixture was partitioned between water (500mL) and EtOAc (500 mL). The aqueous layer was basified with solid Na₂CO₃to pH 11 and filtered through a pad of celite. The aqueous layer andcelite pad were extracted with EtOAc (3×500 mL) and the combinedextracts were washed with saturated aqueous NaHCO₃ solution (500 mL),dried (MgSO₄) and evaporated in vacuo to affordN-(4-(4-aminonaphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide (12)(35.0 g, 78%) as a purple foam: m/z 324 (M+H)⁺ (ES⁺).

N-(4-(4-(3-(3-tert-Butyl-1-D-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(1)

To a stirred suspension of CDI (28.8 g, 178 mmol) in DCM (250 mL) at RT,under nitrogen, was added 3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine (7)(40.7 g, 178 mmol) portion-wise over 1 hr. After 1 hr the resulting darkred solution was added dropwise, over 1 hr, to a stirred solution ofN-(4-(4-aminonaphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide (12)(35.5 g, 99 mmol) in DCM (1 L). After 1 hr MeOH (100 mL) was added andthe mixture was kept at RT for 16 hr. The reaction mixture wasevaporated in vacuo and the residue was taken up into DCM (500 mL) andwas washed with water and saturated aqueous NaHCO₃ solution (500 mL).The organic layer was dried (MgSO₄), evaporated in vacuo and the residuepurified by flash column chromatography (SiO₂, 800 g, 2% MeOH in DCM,isocratic elution). This product was recrystallized from ethylacetate/heptane (800 mL of a 5:3 v/v mixture) to affordN-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(1) (25.5 g, 45%) as a white powder. Found: C, 68.41; H, 5.93; N, 14.36;C₃₃H₃₄N₆O₄ requires: C, 68.49; H, 5.92; N, 14.52%.

Biological Testing

Summaries of the properties of the compound of formula (1) establishedusing in vitro assays are presented below. The compound of formula (1)showed substantial differences in its profiles to BIRB796. Although bothcompounds were potent and effective inhibitors of LPS-induced TNFαrelease in THP-1 cells and differentiated U937 cells (Table 1), BIRB796showed no significant effect (NSE) in the six other systems weinvestigated, namely: LPS-induced IL-8 release in differentiated U937cells (BIRB796 31% of maximum inhibition; the compound of formula (1)IC₅₀: 7.9 nM); LPS-induced IL-8 release from sputum macrophages (Table2); poly I:C induced ICAM1 expression in human bronchial epithelial cellline, BEAS2B cells (BIRB796 no effect at 10 ug/ml; the compound offormula (1) IC₅₀: 1.7 nM), rhinovirus-induced ICAM1 expression in BEAS2Bcells (Table 2); rhinovirus-induced IL-8 release in BEAS2B cells (Table2) and rhinovirus replication in MRC5 cells. In marked contrast, thecompound of formula (1) demonstrated activity in all six systems andshowed levels of potency which were equivalent to or exceeded those indemonstrated in LPS-induced TNFα release in U937 cells.

TABLE 1 Comparative p38 MAP kinase activity and LPS induced TNF- αactivity of macrophages for BIRB796 and Compound (1) IC₅₀ Values (nM)Test p38 MAPK Enzymes LPS-induced TNFα release Compound α subtype γsubtype d-U937 cells^(a) THP1 cells BIRB796 12 (n = 6) 296 (n = 5)  20(n = 2) 12 (n = 3) (1) 12 (n = 2) 344 (n = 2) 2.1 (n = 3) 13 (n = 3)^(a)d-U937 cells = Differentiated U937 cells

TABLE 2 Comparative Anti-Inflammatory Activity of BIRB796 and Compound(1) IC₅₀ Values (nM) BEAS2B cells Sputum macrophages Rhinovirus-Rhinovirus- Test LPS- induced induced ICAM induced IL-8 Compound IL-8release expression release BIRB796 NSE @ 19 μM NSE @ 1.9 μM NSE @ 1.9 μM(n = 2) (n = 2) (n = 2) (1) 5 0.37 0.065 (n = 1) (n = 4) (n = 4)

The potential of these two compounds to inhibit rhinovirus replicationin human fetal lung fibroblast, MRC5 cells has been investigatedfurther. It was established that BIRB796 was without effect on viralreplication over a concentration range up to 1.9 μM. However, weinvestigated the concentration effect curves of the compound of formula(1) and found that a concentration of 5.2 nM decreased the viral titreby 1 log order.

A description of these assays is as follows:

Enzyme Inhibition Assay

The enzyme inhibitory activity of compound was determined by FRET usingsynthetic peptides labelled with both donor and acceptor fluorophores(Z-LYTE, Invitrogen Ltd., Paisley, UK). Recombinant, phosphorylated p38MAPK γ (MAPK12:Millipore) was diluted in HEPES buffer, mixed withcompound at desired final concentrations and incubated for two hours atroom temperature. The FRET peptide (2 μM) and ATP (100 μM) were nextadded to the enzyme/compound mixture and incubated for 1 hr. Developmentreagent (protease) was added for 1 hr prior to detection in afluorescence microplate reader (Varioskan® Flash, ThermoFisherScientific). The site-specific protease cleaves only non-phosphorylatedpeptide and eliminates the FRET signal. Phosphorylation levels of eachreaction were calculated using the ratio of coumarin emission (donor)over fluorescein emission (acceptor) with high ratios indicating highphosphorylation and low ratios, low phosphorylation levels. Thepercentage inhibition of each reaction was calculated relative tonon-inhibited control, and the 50% inhibitory concentration (IC₅₀ value)then calculated from the concentration-response curve.

For p38 MAPK alpha (MAPK14: Invitrogen), enzyme activity was evaluatedindirectly by determining activation/phosphorylation of the down-streammolecule, MAPKAP-K2. The p38 MAPKα protein was mixed with the testcompound for 2 hr at RT. The p38a inactive target MAPKAP-K2 (Invitrogen)and FRET peptide (2 μM), which is a phosphorylation target forMAPKAP-K2, and ATP (10 μM) were then added to the enzymes/compoundmixture and incubated for one hour. Development reagent was then addedand the mixture incubated for one hour before detection by fluorescencecompleted the assay protocol.

LPS-Induced TNF α Release in U937 Cells and THP-1 Cells: Potency

U937 cells, human monocytic cell line, were differentiated tomacrophage-type cells by incubation with phorbol myristate acetate (PMA;100 ng/ml) for 48 to 72 hr. Where appropriate, cells were pre-incubatedwith final concentrations of compound for 2 hr. Cells were thenstimulated with 0.1 μg/ml of LPS (from E. Coli: O111:B4, Sigma) for 4hr, and the supernatant collected for determination of TNFαconcentration by sandwich ELISA (Duo-set, R&D systems). The inhibitionof TNFα production was calculated as a percentage of that achieved by 10μg/ml of BIRB796 at each concentration of test compound by comparisonwith vehicle control. The relative 50% effective concentration (R-EC₅₀)was determined from the resultant concentration-response curve. THP-1,human monocytic cell line, was also used for this assay. THP-1 cellswere stimulated with 3 μg/ml of LPS (from E. Coli: O111:B4, Sigma) for 4hr, and the supernatant collected for determination of TNFαconcentration. The 50% inhibitory concentration (IC₅₀) was determinedfrom the resultant concentration-response curve.

LPS-Induced IL-8 Release in U937 Cells: Potency

U937 cells, human monocytic cell line, were differentiated tomacrophage-type cells by incubation with phorbol myristate acetate (PMA;100 ng/ml) for 48 to 72 hours. Cells were pre-incubated with finalconcentrations of compound for 2 hrs. Cells were then stimulated with0.1 μg/ml of LPS (from E. Coli: O111:B4, Sigma) for 4 hrs, and thesupernatant collected for determination of the IL-8 concentration bysandwich ELISA (Duo-set, R&D systems). The inhibition of IL-8 productionwas calculated at each concentration of test compound by comparison withvehicle control. The 50% inhibitory concentration (IC₅₀) was determinedfrom the resultant concentration-response curve.

Poly I:C-Induced ICAM-1 Induction in BEAS2B Cells: Potency

Poly I:C (1 ug/ml) (Invivogen Ltd., San Diego, Calif.) was transfectedinto BEAS2B cells (human bronchial epithelial cells, ATCC) withOligofectamine (Invitrogen, Carlsbad, Calif.). Cells were pre-incubatedwith final concentrations of compound for 2 hrs. The level of ICAM1expression on the cell surface was determined by cell-based ELISA.Briefly, at 18 hrs after poly I:C transfection, cells were fixed with 4%formaldehyde in PBS. After quenching endogenous peroxidase by adding0.1% sodium azide and 1% hydrogen peroxide, cells were washed withwash-buffer (0.1% Tween in PBS: PBS-Tween). After blocking the wellswith 5% milk in PBS-Tween for 1 hr, the cells were incubated withanti-human ICAM-1 antibody (Cell Signaling Technology, Danvers, Mass.)in 1% BSA PBS overnight at 4° C. Cells were washed with PBS-Tween andincubated with the secondary antibody (HRP-conjugated anti-rabbit IgG,Dako Ltd., Glostrup, Denmark). The ICAM-1 signal was detected by addingsubstrate and reading the absorbance at 450 nm with a referencewavelength of 655 nm using a spectrophotometer. The cells were thenwashed with PBS-Tween and total cell numbers in each well weredetermined by reading absorbance at 595 nm after Crystal Violet stainingand elution by 1% SDS solution. The measured OD 450-655 readings werecorrected for cell number by dividing with the OD595 reading in eachwell. The inhibition of ICAM-1 expression was calculated at eachconcentration of test compound by comparison with vehicle control. The50% inhibitory concentration (IC₅₀) was determined from the resultantconcentration-response curve.

Sputum Macrophage Assay

Sputum was induced by inhalation of a nebulised solution of 3% (w/v)hypertonic saline to healthy volunteers. Dithiothreitol (0.02% at final)was then added and mixed vigorously using a vortex mixer until thesputum became less viscous. The cell pellet produced by centrifugation(at 1500 rpm for 10 min) was resuspended in 10% FCS RPMI-1640, andsputum macrophages were separated by plate adhesion in high bindingplate (CellBIND®, Corning Limited. UK) for 2 hr. The adhered cells werewashed with RPMI-1640, and stimulated with LPS (1 ug/ml). After 4 hrincubation, the supernatant was collected for measurement of IL-8production using Duoset ELISA development kit (R&D systems, Minneapolis,Minn.). Compounds were added 2 hr before LPS stimulation.

Rhinovirus-Induced IL-8 and ICAM-1

Human rhinovirus RV16 (HRV) was obtained form American Type CultureCollection (Manassas, Va.). Viral stocks were generated by infectingHela cells with HRV until 80% of the cells were cytopathic.

BEAS2B cells were infected with 5 MOI (multiplicity of infection of 5)of HRV and incubated for 2 hr at 33° C. with gentle shaking forabsorption. The cells were then washed with PBS, fresh media added andthe cells were incubated for a further 72 hr. The supernatant wascollected for assay of IL-8 concentrations using Duoset ELISAdevelopment kit (R&D systems, Minneapolis, Minn.).

The level of ICAM-1 expressing cell surface was determined by cell-basedELISA. After appropriate incubation, cells were fixed with 4%formaldehyde in PBS. After quenching endogenous peroxidase by adding0.1% sodium azide and 1% hydrogen peroxide, wells were washed withwash-buffer (0.05% Tween in PBS: PBS-Tween). After blocking well with 5%milk in PBS-Tween for 1 hr, the cells were incubated with anti-humanICAM-1 antibody in 5% BSA PBS-Tween (1:500) overnight. Wells were washedwith PBS-Tween and incubated with the secondary antibody (HRP-conjugatedanti-rabbit IgG, Dako Ltd.). The ICAM-1 signal was detected by addingsubstrate and reading at 450 nm with a reference wavelength of 655 nmusing a spectrophotometer. The wells were then washed with PBS-Tween andtotal cell numbers in each well were determined by reading absorbance at595 nm after Crystal Violet staining and elution by 1% SDS solution. Themeasured OD₄₅₀₋₆₅₅ readings were corrected for cell number by dividingwith the OD₅₉₅ reading in each well. Compounds were added 2 hr beforeHRV infection and 2 hr after infection when non-infected HRV was washedout.

Rhinovirus-Titre Assay

MRC5 cells (human lung fibroblast, ATCC) were infected with 1 MOI(multiplicity of infection of 1.0) of HRV and incubated for 1 hr at 33°C. with gentle shaking for absorption. The cells were then washed withPBS, fresh media added and the cells were incubated for a further 96 hr.The supernatant was collected and 10-fold serial dilutions of viruscontaining supernatant were prepared. All titrations were performed byinfecting confluent Hela cell monolayers with serially dilutedsupernantant (10⁻¹ to 10⁻⁵) and assessing cytopathetic effect by MTTassay 4 days after infection. The amount of virus required to infect 50%of Hela cells was calculated in each treatment as TCID₅₀ U/mL. Compoundswere added 24 and 2 hr before HRV infection and 1 hr after infectionwhen non-infected HRV was washed out.

LPS-Induced Neutrophils Accumulation in Mice

Non-fasted mice were dosed by the intra tracheal route with eithervehicle, or the test substance at the indicated times (within the range2-12 hr) before starting LPS treatment. At T=0, mice were placed into anexposure chamber and exposed to LPS. Eight hours after LPS challenge,animals were under anesthetized, the trachea cannulated and BALFextracted by infusing and withdrawing 1 ml of PBS into the lungs via atracheal catheter. Total and differential white cell counts in the BALFsamples were measured using a Neubaur haemocytometer. Cytospin smears ofthe BALF samples were prepared by centrifugation at 200 rpm for 5 min atroom temperature and stained using a DiffQuik stain system (DadeBehring). Cells were counted using oil immersion microscopy. Treatmentof mice with Compound (1) was found to inhibit neutrophil accumulationinto the BALF when treated at 2, 8 or 12 hr before LPS challenge (Table3 and 4)

TABLE 3 Effects of treatment with Compound (1) Treatment Neutrophilnumbers in BAL (×10⁵/mL) mg/ml Compound (1) 2 hr pre-dose 12 hr pre-doseVehicle 20.2 ± 3.7 —  0.02 15.1 ± 2.1 20.1 ± 2.9 0.1 10.4 ± 1.6 16.7 ±2.4 0.2  4.6 ± 1.2 14.3 ± 2.0 Results are presented as the mean ± SEM, n= 8

TABLE 4 Effects of treatment with Compound (1) Neutrophil numbers in BAL(×10⁵/mL) Treatment 8 hr pre-dose Vehicle 16.38 ± 2.53 Compound (1) 9.65 ± 1.50 0.2 mg/ml Results are presented as the mean ± SEM, n = 8Allergen-Induced Eosinophil Accumulation in Guinea Pigs

Dunkin Hartley guinea pigs were immunized with ovalbumin. Six doses ofvehicle or Example 8 (1.5 mg/ml) were administered by aerosol every 12hours with the final dose being administered 2 hr before initiating ofthe allergen challenge (grade V, OVA; 10 μg/mL solution aerosolisedusing a De Vibliss ultrasonic nebuliser 2000, over a 30 min period). Twogroups of animals received 6 doses of Example 8 whilst a further twogroups received 6 doses of vehicle. 8 or 24 hours after OVA challenge(see group details above), the trachea was cannulated and BALFextracted. The procedure for this involved aspirating 5 ml of PBS intothe lungs via a tracheal catheter. Total and differential white cellcounts in the BAL fluid samples were measured using a Neubaurhaemocytometer. Cytospin smears of the BAL fluid samples were preparedby centrifugation at 200 rpm for 5 min at room temperature and stainedusing a DiffQuik stain system (Dade Behring). Cells were counted blindusing oil immersion microscopy.

Treatment of guinea pigs with Compound (1) was found to inhibiteosinophil accumulation into the BALF when investigated at 8 and 24 hrpost ovalbumin challenge (Table 5)

TABLE 5 Inhibition of eosinophils in BALF following allergen challengeNeutrophil numbers in BAL (×10⁵/mL) Treatment 2 hr pre-dose 12 hrpre-dose Compound(1) 12.4 ± 1.7 21.6 ± 3.9 Results are presented as themean ± SEM, n = 6Cigarette Smoke Model

A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4%cigarette smoke, diluted with compressed air) for 30 min/day for 11 daysusing a Tobacco Smoke Inhalation Experiment System for small animals(Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). Testsubstances were given intra-nasally (35 μl of solution in 50% DMSO/PBS)and therapeutically twice daily for 3 days after the final cigarettesmoke exposure. Twelve hours after the last dosing, animals wereanesthetized, the trachea cannulated and bronchoalveolar lavage fluid(BALF) was collected. The numbers of alveolar macrophages andneutrophils were determined by FACS analysis (EPICS® ALTRA II, BeckmanCoulter, Inc., Fullerton, Calif., USA) using anti-mouse MOMA2 antibody(macrophage) or anti-mouse 7/4 antibody (neutrophil).

The results of treatment with Compound (1) are shown in FIG. 1(neutrophils) and FIG. 2 (activated alveolar macrophages). Data for cellnumbers are shown as the mean±SEM.

The cigarette smoke model used for this study is reported as acorticosteroid refractory system, (Medicherla S. et al., (2008); J.Pharmacol. Exp. Ther. 324(3):921-9) and it was confirmed thatfluticasone propionate did not inhibit either neutrophil or macrophageaccumulation into airways at 50 g/mL (35 μl, bid, in), the same dosethat produced >80% inhibition of LPS-induced neutrophil accumulation.However, treatment with Compound (1) was found to result in a marked,dose-dependent reduction in the numbers of both neutrophils andactivated macrophages.

Ovalbumin Challenge/Parainfluenza Infection Model

Male Dunkin-Hartley guinea-pigs (300-350 g, n=6/group) were sensitisedwith 100 μg ovalubumin (OVA)+100 mg Al₂(OH)₃ in 1 ml normal saline(i.p.) on days 2 and 6. Parainfluenza virus (PIV-3; 10⁶ infectiousunits) or media without virus was nasally instilled on days 11 and 12.Animals were treated with either nebulised (i) fluticasone propionate ata dose of 1.5 mg per day (initial studies established that this dose offluticasone propionate inhibited ovalbumin-mediated lung functionchanges in sensitized animals treated with PIV3 medium) or (ii) Compound(1) (0.15 mg per day) or (iii) a combination of fluticasone propionateand Compound (1) at the doses indicated above or (iv) the vehicle(DMSO:ethanol:saline, 30:30:40%) from days 10-15. All animals werechallenged for 1 hr with nebulised OVA (10 μg/ml) on day 15 and repeatedmeasurements of specific airways conductance (sG_(aw)) were made over a24 h period using whole body plethysmography. Measurements of sG_(aw)after OVA challenge are plotted as % change from baseline. FIG. 3 showsthe effect of Compound 1 as monotherapy, while FIG. 4 shows its effectswhen administered in combination with fluticasone propionate. Treatmentwith Compound (1) alone was found to produce no effect on the initial(1^(st) hr) bronchoconstrictor response to ovalbumin challenge, butmarkedly inhibited the subsequent response (2-12 hr post treatment).When co-administered with fluticasone propionate, both the initial andsubsequent bronchoconstrictor responses evoked by ovalbumin challengewere inhibited.

SUMMARY

The biological studies in vitro show that the compound of formula (1) isa potent inhibitor of p38 MAP kinase subtypes alpha and gamma with goodefficacy in an in vitro model of anti-inflammatory activity (LPS-inducedTNFα release from differentiated U937 cells and THP-1 cells). Inaddition, the compound of formula (1) shows the surprising properties ofbeing able to inhibit both rhinovirus-induced inflammation andrhinovirus replication

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the claims.

The invention claimed is:
 1. A method of treatment of respiratory viralinfections in patients comprising administering to a subject in needthereof an effective amount of the compound of the formula:

or a pharmaceutically acceptable salt thereof, including all stereoisomers and tautomers thereof.
 2. The method of claim 1 furthercomprising administering an anti-viral therapy selected from zanamavir,oseltamivir and oseltamivir phosphate.
 3. The method of claim 1 whereinthe respiratory viral infection is in a patient with one or more chronicconditions selected from congestive heart failure, diabetes, and cancer.4. The method of claim 1 wherein the respiratory viral infection is inan immunosuppressed patient.
 5. The method of claim 1 wherein thecompound is administered in combination with one or morepharmaceutically acceptable diluents or carriers.
 6. A method oftreatment of respiratory viral infections in patients with chronicconditions selected from congestive heart failure, diabetes, cancer, orin immunosuppressed patients, said method comprising administering to asubject in need thereof an effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof, including all stereoisomers and tautomers thereof: